- Email: [email protected]
The effect of PhTx3 on the release of 3H-acetylcholine induced by tityustoxin and potassium in brain cortical slices and myenteric plexus
NEUROSglENC[ ELSEVIER
Neuroscienee Letters 196 (1995) 131-133
[HT[IS
The effect of PhTx 3 on the release of 3H-acetylcholine induced by tityustoxin and potassium in brain cortical slices and myenteric plexus R . S . G o m e z a, T . A . A . C a s a l i a, M . A . R o m a n o - S i l v a a, M . N . C o r d e i r o c, C . R . D i n i z c , T. M o r a e s - S a n t o s b, M . A . M . P r a d o a, M . V . G o m e z ~,* aDepartamento de Farmacologia, lnstituto de O~ncias Biol6gicas, UFMG, Caixa Postal 2486, 30161-970 Belo Horizonte, Minas Gerais, Brazil bDepartamento de Alimentos, Faculdade de Farmd~ia, UFMG , Caixa Postal 2486, 30161-970 Belo Horizonte, Minas Gerais, Brazil CFUNED, Belo Horizonte, Minas Gerais, Brazil
Received 30 March 1995; revised version received 7 July 1995; accepted 9 July 1995
Abstract
The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca2÷-dependent glutamate release and the increase in cytosolic free Ca2÷ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KC1 in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extraceUular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.
Keywords: Tityustoxin; Acetylcholine; Myenteric plexus; Brain cortical slices; Calcium channels; Transmitter release; Phoneutria nigriventer
It has been established that depolarization of nerve terminals increases Ca 2÷ permeability, and the subsequent Ca 2+ influx by voltage-sensitive calcium channels (VSCCs) promotes the release of neurotransmitter in the synapse. Based on pharmacological and biophysical data, neuronal VSCCs have been classified into several subtypes, including L, N, T and P [4,13]. The venom of the Brazilian spider Phoneutria nigriventer possesses several toxic polypeptidic fractions, some of which are neurotoxic [15]. One of the toxic components, designated PhTx 3, suppressed Ca2÷-dependent glutamate release and the increase in cytosolic free Ca 2÷ that occurred in response to membrane depolarization by KCI (Prado et al., submitted), suggesting that PhTx 3 might be a novel neuro~aal Ca 2÷ channel blocker. In this work, we investigated the effect of PhTx 3 on the release of acetylcholine (ACh) from cerebral cortex and peripheral neurons.
* Corresponding author, Fax: +55 31 4415963.
Tityustoxin (TsTX), an a-scorpion toxin that activates voltage-sensitive sodium channels and uptake of calcium in synaptosomes [10], is useful as a tool to study the release of neurotransmitters [6,8,]. Recently, the calcium channels involved with the release of ACh induced by TsTX in brain have been pharmacologically characterized, making it an attractive way to evoke the release of neurotransmitter [ 1]. TsTX was purified as previously described [7] and PhTx 3 was purified according to Cordeiro et al. [2]. toAgaIVA was obtained from Peptide (Osaka, Japan). All other chemicals were of analytical grade. Wistar rats (200-250 g) of either sex were killed by decapitation and their cerebral cortices were removed and sliced in a Mcllwain Tissue Slicer (Brinckman Instruments Inc., UK). Pieces of longitudinal muscle with its accompanying plexus were obtained from guinea-pigs of either sex, as described by Rang [14]. The release of ACh into the incubating medium was studied after labelling tissue ACh with methyl [3H]-choline chloride (78 Ci/mmole, Amersham Searle), as previously described [9].
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(95)1 1843-I
132
R.S. Gomez et al. / Neuroscience Letters 196 (1995) 131-133
Table 1 Effect of PhTx 3 and EGTA on the release of ACh induced by TsTX and KCI in guinea-pig myenteric plexus [3H]-ACh (dpm/mg of tissue)
None EGTA (2.0 mM) PhTx 3 (1.0#g/ml)
Control
TsTX (2.5 #M)
KCI (50 mM)
53.0±4.5 54.3 ± 4.4 48.3 .+ 2.0
130.9_+1.3' 61.8 ,+ 4.4** 65.1 ± 1.2"*
114.7,+7.5" 55.3 .+ 1.2"* 61.4 _+ 1.5'*
Pieces of longitudinal muscle with its accompanying plexus (-+40 mg) were pre-incubated for 15 min in Krebs/trizma medium in the absence (control) or in the presence of PhTx 3 (1.0 /.tg/ml) or EGTA (2.0 raM). They were then stimulated for 30 min with tityustoxin (2.5/~M) or KCI (50 mM). The values express the mean ± SEM for duplicates from at least three different experiments. *Statistically different from the control value, P < 0.05. **Statistically different from the tityustoxin or KCI value, P < 0.05.
In order to characterize the radioactivity released, the supernatants of a small number of experiments were submitted to high-voltage electrophoresis to separate [3H]-choline, and [3H]-acetylcholine or ACh was separated from choline by the choline kinase method [5]. [3H]-acetylcholine represented 60-72% of the total radioactivity. Statistics was performed by Analysis of Variance (ANOVA). Table 1 shows the effect of PhTx 3 and 2.0 mM EGTA (no calcium added in the medium) on the release of ACh induced by TsTX and K÷ in the myenteric plexus longitudinal muscles of guinea-pig ileum. In the presence of EGTA (2.0 mM) the release of ACh induced by TsTX and K÷ was markedly reduced (P < 0.05). Thus, in the myenteric plexus, the stimulated release of ACh is strictly dependent on the presence of extracellular Ca 2+. The PhTx 3 had a very pronounced effect in the myenteric plexus, inhibiting ACh release induced by both stimuli; the values obtained in this experiment were similar to those obtained in the presence of EGTA (P > 0.05). Table 2 shows a similar experiment performed in brain cortical slices. In the presence of 2.0 mM EGTA (no cal-
cium added to the medium), the evoked release of ACh by TsTX and KC1 was again abolished. The Phoneutria nigriventer toxic component, PhTx3 (1.0/tg/ml), inhibited by 65.5 _ 1.7% and 57.1 __.5.9%, respectively, the release of ACh induced by TsTX and KC1 from brain slices, and the extent of the inhibition was significantly smaller than that obtained in the presence of EGTA. The main calcium channel involved in the release of ACh induced by TsTX and KC1 from brain slices is sensitive to low concentration of ~o-AgalVA, a P type calcium channel blocker [1]. Thus, we investigated whether we could inhibit this residual Ca2+-dependent release induced by TsTX and 50 mM KCI by associating PhTx3 and to-AgalVA. In similar conditions as described above, ta-AgatoxinlVA has been shown to inhibit by 51 ___4.2% the release of ACh induced by TsTX in rat cortical slices [1]. In our experiments, toAgalVA inhibited by 55.8 _+3.2% the release of ACh evoked by K+ (50 mM) in rat cortical slices (data not shown) in agreement with previous data [1]. The association of ~o-AgalVA with PhTx3 did not abolish the release of ACh from brain cortical slices stimulated by TsTX or KCI. Hence, the amount of transmitter released was not statistically different from that in the experiments where slices were treated only with PhTx3. Therefore, there was no additive inhibitory effect in the release of ACh when both toxins were associated. PhTx 3 had a clear effect inhibiting the increase of intracellular Ca 2+ in depolarized synaptosomes, and consequently this toxic fraction blocked calcium-dependent release of glutamate from synaptosomes (Prado et. al., submitted). This effect occurs most likely due to the interference with one or more calcium channels. The release of ACh by TsTX is Na +- and Ca2+-dependent and it is inhibited by tetrodotoxin [6,8]. TsTX causes membrane depolarization that probably opens calcium channels allowing the influx of Ca 2÷ into rat brain cortical slices [8] and synaptosomes [10,16]. As the release of ACh by TsTX and K ÷ is calcium-dependent, we asked whether the target of PhTx 3 could be involved in supplying Ca 2+ to release ACh in the central and peripheral nervous system.
Table 2 Effect of PhTx3, EGTA and ~o-Aga-IVA on the release of ACh induced by TsTX and KCI in rat brain cortical slices [3H]-ACh (dpm/mg of tissue)
None EGTA (2.0 raM) PhTx3 (1.0/tg/ml) PhTx3 (1.0/~g/ml) plus ~-AgalVA (0.1/~M)
Control
TsTX (2.5 #M)
KC1 (50 mM)
98.2 ,+ 2.7 93.2 ± 3.8 104.4 .+ 6.0
196.6 ± 2.5* 102.0 ± 3.9** 133.4 ± 3.0 **,+
187.2 ,+ 2.1" 112.9 ± 4.1"* 137.2 _+6.3 **'+
102.2 ± 3.5
132.1 ± 2.7 **,+
150.0 _+ 11.48 **'+
Brain cortical slices (.+ 40 mg) were pre-incubated for 15 min in Krebs/trizma medium in the absence (control) or in the presence of PhTx3 (I.0 #g/ml), EGTA (2.0 mM), or PhTx3 (l.0#g/ml) plus to-AgalVA (0.1/~M). The values express the mean _+SEM for duplicates from at least three different experiments. *Statistically different from the control value, P < 0.05. **Statistically different from the tityustoxin or KCI value, P < 0.05. +Statistically different from the values obtained in the presence of EGTA.
R.S. Gomez et al. / Neuroscience Letters 196 (1995) 131-133
PhTx 3 decreased the release o f A C h induced by TsTX and 50 m M K + in brain cortical slices and myenteric plexus. The inhibitory effect o f PhTx 3 was greater in the myenteric plexus than in the brain, suggesting that the target o f PhTx 3 allows the passage of most of the Ca 2÷ involved with the release o f ACh in the myenteric plexus. It is also possible that PhTx 3 has more than one target, interacting with different kinds of calcium channels in central and peripheral neurons. In fact, the results obtained in the myenteric plexus and the association of PhTx 3 and ~o-AgalVA in brain slices support this notion. Thus, while in corticail slices co-AgalVA is able to partially block the stimulated release of [3H]ACh [1], it could not alter the residual release of [3H]ACh that was insensitive to PhTx 3. These results might suggest that PhTx 3 could interact with co-AgaIVA sensitive calcium channels. However, P type channels do not seem to have a role in neurotransmitter release from the myenteric plexus [12], and co-AgalVA was unable to block the release of ACh stimulated by TsTX or KC1 in the myenteric plexus (unpublished results). Indeed, N type channels are considered the main pathway for calcium entry in these nerve terminals [3,11,17]. Since PhTx 3 was very effective at blocking ACh release in the myenteric plexus, but did not have an additive effect with co-AgalVA in brain cortical slices, we suggest that this toxin may interact with more than one of the known calcium channels. The PhTx 3 is composed o f six polypeptides (Tx3-1 to Tx3-6) with weak h o m o l o g y among themselves and molecular weights ranging from 3500 to 8500 D a . Only one peptide, designated Tx3-3 with molecular weight of 6300 Da, interferes with intracellular calcium increase due to membrane depolarization and is able to abolish the CaZ+-dependent release o f glutamate (Prado et al., submitted). This peptide is able to reproduce quantitatively the effect o f PhTx 3 on inlracellular Ca 2+, glutamate release (Prado et al., submitted) and also ACh release (data not shown). The TX3-3 N-terminal sequence shows very weak homologies with other spider toxins that block calcium channels [2], making it a putative new tool to investigate these important cellular components. In summary, we concluded that extracellular Ca 2÷ is essential for the stimuli-induced ACh release from brain cortical slices and guinea-pig myenteric plexus and that the target o f PhTx 3 takes part in the process of ACh release in brain and autonomic nervous system. W e thank A d r i a n e A. Pereira and Antonio C.S. Gomes for technical assistance, and Dr. L.A. De Marco for reading and suggestions in this manuscript. This work was supported by grants of Finep, CNPq, Fapemig, PRPqU F M G and an I S N Seed Grant to M.A.M.P.
133
[1] Casali, T.A.A., Gomez, R.S., Moraes-Santos, T. and Gomez, M.V., Differential effects of calcium channel antagonists on tityustoxin and ouabain induced release of 3H-Acetylcholin¢ from brain cortical slices, Neuropharmacology, (1995) in press. [2] Cordeiro, M.N., Figueiredo, S.G., Valentim, A.C., Diniz, C.R., Von Eickstedt, V.R.D., Gilroy, J. and Richardson, M., Purification and amino acid sequence of six Tx3 type neurotoxins from the venom of the Brazilian "armed" spider Phoneutria nigriventer, Toxicon, 31(I) (1993) 35-42. [3] De Luca, A., Li, C.G., Rand, M.J., Reid, J.J., Thaina, P. and Wong-Dusting, H.K., Effects of co-conotoxinGVIA on autonomic neuroeffector transmission in various tissues, Br. J. Pharmacol., 101 (1990) 437-447. [4] Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons, J. Physiol., 394 (1987) 149-172. [5] Goldberg, A.M. and McCaman, R.E., The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem, 20 (1973) 1-8. [6] Gomez, M.V., Dai, M.E.M. and Diniz, C.R., Effects of scorpion venom tityustoxin on the release of acetylcholine from incubated slices of rat brain, J. Neurochem., 20 ( 1973) 1051-1061. [7] Gomez, M.V. and Diniz, C.R., Separation of toxic components from the Brazilian scorpion Tityus serrulatus venom, Mem. Inst. Butantan Symp. Int., 33 (1966) 899-902. [8] Gomez,M.V., Diniz, C.R. and Barbosa, T.S., A comparison of the effect of scorpion venom tityustoxin and ouabaln on the release of acetylcholinc from incubated slices of rat brain, J. Neurochem., 24 (1975) 331-336. [9] Gomez, R.S., Moraes-Santos, T. and Gomez, M.V., Effect of lanthanum ions on the release of acetylcholine induced by tityustoxin, K+ and ouabain from myenteric plexus and brain cortical slices, Toxicon, 31 (1993) 411-415. [10] Henriques, M.C. and Gomez, M.V., The effect of scorpion venom tityustoxin on the uptake of calcium in synaptosomes, Brain Res. Bull, (1981) 255-259. [11] Lundy, P.M. and Frew, R., Evidence of o~-conotoxin GVIAsensitive Ca2+ channels in mammalian peripheral nerve terminals, Eur. J. Pharmacol., 231(1988) 325-330. [12] Lundy, P.M. and Frew, R., Effect of w-agatoxin-IVA on autonomic neurotransmission, Eur. J. Pharmacol., 261 (1994) 79-84. [13] Mintz, I.M., Venema, V.J., Swiderek, K.M., Lee, T.D., Bean, B.P and Adams, M.E., P-Type Ca2+ channels blocked by the spider toxin co-Aga-IVA,Nature, 335 (1992) 827-829. [14] Rang, H.P., Stimulant actions of volatile anaesthetics on smooth muscle, Brit. J. Pharmacol., 22 (1964) 356-365. [15] Rezende, L., Jr., Cordeiro, M.N., Oliveira, E.B. and Diniz, C.R., Isolation of neurotoxic peptides from the venom of the 'armed' spider Phoneutria nigriventer, Toxicon, 29 (1991) 1225-1233. [16] Romano-Silva, M.A., Gomez, M.V. and Brammer, M.J., Modulation of Ca2+-stimulated glutamate release from synaptosomes by Na+ entry through tetrodotoxin-sensitive channels, Biochem. J., 304 (1994) 353-357. [17] Wessler, I., Dooley, D.J., Werland, J. and Sclemmer, F., Differential effects of calcium channel antagonists (w-conotoxin GVIA, nifedipine, verapamil) in the electrically-evoked release of [3H]acetylcholine from the myenteric plexus, phrenic nerve and neocortex of rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 341 (1990) 288-294.
Neuroscienee Letters 196 (1995) 131-133
[HT[IS
The effect of PhTx 3 on the release of 3H-acetylcholine induced by tityustoxin and potassium in brain cortical slices and myenteric plexus R . S . G o m e z a, T . A . A . C a s a l i a, M . A . R o m a n o - S i l v a a, M . N . C o r d e i r o c, C . R . D i n i z c , T. M o r a e s - S a n t o s b, M . A . M . P r a d o a, M . V . G o m e z ~,* aDepartamento de Farmacologia, lnstituto de O~ncias Biol6gicas, UFMG, Caixa Postal 2486, 30161-970 Belo Horizonte, Minas Gerais, Brazil bDepartamento de Alimentos, Faculdade de Farmd~ia, UFMG , Caixa Postal 2486, 30161-970 Belo Horizonte, Minas Gerais, Brazil CFUNED, Belo Horizonte, Minas Gerais, Brazil
Received 30 March 1995; revised version received 7 July 1995; accepted 9 July 1995
Abstract
The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca2÷-dependent glutamate release and the increase in cytosolic free Ca2÷ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KC1 in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extraceUular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.
Keywords: Tityustoxin; Acetylcholine; Myenteric plexus; Brain cortical slices; Calcium channels; Transmitter release; Phoneutria nigriventer
It has been established that depolarization of nerve terminals increases Ca 2÷ permeability, and the subsequent Ca 2+ influx by voltage-sensitive calcium channels (VSCCs) promotes the release of neurotransmitter in the synapse. Based on pharmacological and biophysical data, neuronal VSCCs have been classified into several subtypes, including L, N, T and P [4,13]. The venom of the Brazilian spider Phoneutria nigriventer possesses several toxic polypeptidic fractions, some of which are neurotoxic [15]. One of the toxic components, designated PhTx 3, suppressed Ca2÷-dependent glutamate release and the increase in cytosolic free Ca 2÷ that occurred in response to membrane depolarization by KCI (Prado et al., submitted), suggesting that PhTx 3 might be a novel neuro~aal Ca 2÷ channel blocker. In this work, we investigated the effect of PhTx 3 on the release of acetylcholine (ACh) from cerebral cortex and peripheral neurons.
* Corresponding author, Fax: +55 31 4415963.
Tityustoxin (TsTX), an a-scorpion toxin that activates voltage-sensitive sodium channels and uptake of calcium in synaptosomes [10], is useful as a tool to study the release of neurotransmitters [6,8,]. Recently, the calcium channels involved with the release of ACh induced by TsTX in brain have been pharmacologically characterized, making it an attractive way to evoke the release of neurotransmitter [ 1]. TsTX was purified as previously described [7] and PhTx 3 was purified according to Cordeiro et al. [2]. toAgaIVA was obtained from Peptide (Osaka, Japan). All other chemicals were of analytical grade. Wistar rats (200-250 g) of either sex were killed by decapitation and their cerebral cortices were removed and sliced in a Mcllwain Tissue Slicer (Brinckman Instruments Inc., UK). Pieces of longitudinal muscle with its accompanying plexus were obtained from guinea-pigs of either sex, as described by Rang [14]. The release of ACh into the incubating medium was studied after labelling tissue ACh with methyl [3H]-choline chloride (78 Ci/mmole, Amersham Searle), as previously described [9].
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(95)1 1843-I
132
R.S. Gomez et al. / Neuroscience Letters 196 (1995) 131-133
Table 1 Effect of PhTx 3 and EGTA on the release of ACh induced by TsTX and KCI in guinea-pig myenteric plexus [3H]-ACh (dpm/mg of tissue)
None EGTA (2.0 mM) PhTx 3 (1.0#g/ml)
Control
TsTX (2.5 #M)
KCI (50 mM)
53.0±4.5 54.3 ± 4.4 48.3 .+ 2.0
130.9_+1.3' 61.8 ,+ 4.4** 65.1 ± 1.2"*
114.7,+7.5" 55.3 .+ 1.2"* 61.4 _+ 1.5'*
Pieces of longitudinal muscle with its accompanying plexus (-+40 mg) were pre-incubated for 15 min in Krebs/trizma medium in the absence (control) or in the presence of PhTx 3 (1.0 /.tg/ml) or EGTA (2.0 raM). They were then stimulated for 30 min with tityustoxin (2.5/~M) or KCI (50 mM). The values express the mean ± SEM for duplicates from at least three different experiments. *Statistically different from the control value, P < 0.05. **Statistically different from the tityustoxin or KCI value, P < 0.05.
In order to characterize the radioactivity released, the supernatants of a small number of experiments were submitted to high-voltage electrophoresis to separate [3H]-choline, and [3H]-acetylcholine or ACh was separated from choline by the choline kinase method [5]. [3H]-acetylcholine represented 60-72% of the total radioactivity. Statistics was performed by Analysis of Variance (ANOVA). Table 1 shows the effect of PhTx 3 and 2.0 mM EGTA (no calcium added in the medium) on the release of ACh induced by TsTX and K÷ in the myenteric plexus longitudinal muscles of guinea-pig ileum. In the presence of EGTA (2.0 mM) the release of ACh induced by TsTX and K÷ was markedly reduced (P < 0.05). Thus, in the myenteric plexus, the stimulated release of ACh is strictly dependent on the presence of extracellular Ca 2+. The PhTx 3 had a very pronounced effect in the myenteric plexus, inhibiting ACh release induced by both stimuli; the values obtained in this experiment were similar to those obtained in the presence of EGTA (P > 0.05). Table 2 shows a similar experiment performed in brain cortical slices. In the presence of 2.0 mM EGTA (no cal-
cium added to the medium), the evoked release of ACh by TsTX and KC1 was again abolished. The Phoneutria nigriventer toxic component, PhTx3 (1.0/tg/ml), inhibited by 65.5 _ 1.7% and 57.1 __.5.9%, respectively, the release of ACh induced by TsTX and KC1 from brain slices, and the extent of the inhibition was significantly smaller than that obtained in the presence of EGTA. The main calcium channel involved in the release of ACh induced by TsTX and KC1 from brain slices is sensitive to low concentration of ~o-AgalVA, a P type calcium channel blocker [1]. Thus, we investigated whether we could inhibit this residual Ca2+-dependent release induced by TsTX and 50 mM KCI by associating PhTx3 and to-AgalVA. In similar conditions as described above, ta-AgatoxinlVA has been shown to inhibit by 51 ___4.2% the release of ACh induced by TsTX in rat cortical slices [1]. In our experiments, toAgalVA inhibited by 55.8 _+3.2% the release of ACh evoked by K+ (50 mM) in rat cortical slices (data not shown) in agreement with previous data [1]. The association of ~o-AgalVA with PhTx3 did not abolish the release of ACh from brain cortical slices stimulated by TsTX or KCI. Hence, the amount of transmitter released was not statistically different from that in the experiments where slices were treated only with PhTx3. Therefore, there was no additive inhibitory effect in the release of ACh when both toxins were associated. PhTx 3 had a clear effect inhibiting the increase of intracellular Ca 2+ in depolarized synaptosomes, and consequently this toxic fraction blocked calcium-dependent release of glutamate from synaptosomes (Prado et. al., submitted). This effect occurs most likely due to the interference with one or more calcium channels. The release of ACh by TsTX is Na +- and Ca2+-dependent and it is inhibited by tetrodotoxin [6,8]. TsTX causes membrane depolarization that probably opens calcium channels allowing the influx of Ca 2÷ into rat brain cortical slices [8] and synaptosomes [10,16]. As the release of ACh by TsTX and K ÷ is calcium-dependent, we asked whether the target of PhTx 3 could be involved in supplying Ca 2+ to release ACh in the central and peripheral nervous system.
Table 2 Effect of PhTx3, EGTA and ~o-Aga-IVA on the release of ACh induced by TsTX and KCI in rat brain cortical slices [3H]-ACh (dpm/mg of tissue)
None EGTA (2.0 raM) PhTx3 (1.0/tg/ml) PhTx3 (1.0/~g/ml) plus ~-AgalVA (0.1/~M)
Control
TsTX (2.5 #M)
KC1 (50 mM)
98.2 ,+ 2.7 93.2 ± 3.8 104.4 .+ 6.0
196.6 ± 2.5* 102.0 ± 3.9** 133.4 ± 3.0 **,+
187.2 ,+ 2.1" 112.9 ± 4.1"* 137.2 _+6.3 **'+
102.2 ± 3.5
132.1 ± 2.7 **,+
150.0 _+ 11.48 **'+
Brain cortical slices (.+ 40 mg) were pre-incubated for 15 min in Krebs/trizma medium in the absence (control) or in the presence of PhTx3 (I.0 #g/ml), EGTA (2.0 mM), or PhTx3 (l.0#g/ml) plus to-AgalVA (0.1/~M). The values express the mean _+SEM for duplicates from at least three different experiments. *Statistically different from the control value, P < 0.05. **Statistically different from the tityustoxin or KCI value, P < 0.05. +Statistically different from the values obtained in the presence of EGTA.
R.S. Gomez et al. / Neuroscience Letters 196 (1995) 131-133
PhTx 3 decreased the release o f A C h induced by TsTX and 50 m M K + in brain cortical slices and myenteric plexus. The inhibitory effect o f PhTx 3 was greater in the myenteric plexus than in the brain, suggesting that the target o f PhTx 3 allows the passage of most of the Ca 2÷ involved with the release o f ACh in the myenteric plexus. It is also possible that PhTx 3 has more than one target, interacting with different kinds of calcium channels in central and peripheral neurons. In fact, the results obtained in the myenteric plexus and the association of PhTx 3 and ~o-AgalVA in brain slices support this notion. Thus, while in corticail slices co-AgalVA is able to partially block the stimulated release of [3H]ACh [1], it could not alter the residual release of [3H]ACh that was insensitive to PhTx 3. These results might suggest that PhTx 3 could interact with co-AgaIVA sensitive calcium channels. However, P type channels do not seem to have a role in neurotransmitter release from the myenteric plexus [12], and co-AgalVA was unable to block the release of ACh stimulated by TsTX or KC1 in the myenteric plexus (unpublished results). Indeed, N type channels are considered the main pathway for calcium entry in these nerve terminals [3,11,17]. Since PhTx 3 was very effective at blocking ACh release in the myenteric plexus, but did not have an additive effect with co-AgalVA in brain cortical slices, we suggest that this toxin may interact with more than one of the known calcium channels. The PhTx 3 is composed o f six polypeptides (Tx3-1 to Tx3-6) with weak h o m o l o g y among themselves and molecular weights ranging from 3500 to 8500 D a . Only one peptide, designated Tx3-3 with molecular weight of 6300 Da, interferes with intracellular calcium increase due to membrane depolarization and is able to abolish the CaZ+-dependent release o f glutamate (Prado et al., submitted). This peptide is able to reproduce quantitatively the effect o f PhTx 3 on inlracellular Ca 2+, glutamate release (Prado et al., submitted) and also ACh release (data not shown). The TX3-3 N-terminal sequence shows very weak homologies with other spider toxins that block calcium channels [2], making it a putative new tool to investigate these important cellular components. In summary, we concluded that extracellular Ca 2÷ is essential for the stimuli-induced ACh release from brain cortical slices and guinea-pig myenteric plexus and that the target o f PhTx 3 takes part in the process of ACh release in brain and autonomic nervous system. W e thank A d r i a n e A. Pereira and Antonio C.S. Gomes for technical assistance, and Dr. L.A. De Marco for reading and suggestions in this manuscript. This work was supported by grants of Finep, CNPq, Fapemig, PRPqU F M G and an I S N Seed Grant to M.A.M.P.
133
[1] Casali, T.A.A., Gomez, R.S., Moraes-Santos, T. and Gomez, M.V., Differential effects of calcium channel antagonists on tityustoxin and ouabain induced release of 3H-Acetylcholin¢ from brain cortical slices, Neuropharmacology, (1995) in press. [2] Cordeiro, M.N., Figueiredo, S.G., Valentim, A.C., Diniz, C.R., Von Eickstedt, V.R.D., Gilroy, J. and Richardson, M., Purification and amino acid sequence of six Tx3 type neurotoxins from the venom of the Brazilian "armed" spider Phoneutria nigriventer, Toxicon, 31(I) (1993) 35-42. [3] De Luca, A., Li, C.G., Rand, M.J., Reid, J.J., Thaina, P. and Wong-Dusting, H.K., Effects of co-conotoxinGVIA on autonomic neuroeffector transmission in various tissues, Br. J. Pharmacol., 101 (1990) 437-447. [4] Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons, J. Physiol., 394 (1987) 149-172. [5] Goldberg, A.M. and McCaman, R.E., The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem, 20 (1973) 1-8. [6] Gomez, M.V., Dai, M.E.M. and Diniz, C.R., Effects of scorpion venom tityustoxin on the release of acetylcholine from incubated slices of rat brain, J. Neurochem., 20 ( 1973) 1051-1061. [7] Gomez, M.V. and Diniz, C.R., Separation of toxic components from the Brazilian scorpion Tityus serrulatus venom, Mem. Inst. Butantan Symp. Int., 33 (1966) 899-902. [8] Gomez,M.V., Diniz, C.R. and Barbosa, T.S., A comparison of the effect of scorpion venom tityustoxin and ouabaln on the release of acetylcholinc from incubated slices of rat brain, J. Neurochem., 24 (1975) 331-336. [9] Gomez, R.S., Moraes-Santos, T. and Gomez, M.V., Effect of lanthanum ions on the release of acetylcholine induced by tityustoxin, K+ and ouabain from myenteric plexus and brain cortical slices, Toxicon, 31 (1993) 411-415. [10] Henriques, M.C. and Gomez, M.V., The effect of scorpion venom tityustoxin on the uptake of calcium in synaptosomes, Brain Res. Bull, (1981) 255-259. [11] Lundy, P.M. and Frew, R., Evidence of o~-conotoxin GVIAsensitive Ca2+ channels in mammalian peripheral nerve terminals, Eur. J. Pharmacol., 231(1988) 325-330. [12] Lundy, P.M. and Frew, R., Effect of w-agatoxin-IVA on autonomic neurotransmission, Eur. J. Pharmacol., 261 (1994) 79-84. [13] Mintz, I.M., Venema, V.J., Swiderek, K.M., Lee, T.D., Bean, B.P and Adams, M.E., P-Type Ca2+ channels blocked by the spider toxin co-Aga-IVA,Nature, 335 (1992) 827-829. [14] Rang, H.P., Stimulant actions of volatile anaesthetics on smooth muscle, Brit. J. Pharmacol., 22 (1964) 356-365. [15] Rezende, L., Jr., Cordeiro, M.N., Oliveira, E.B. and Diniz, C.R., Isolation of neurotoxic peptides from the venom of the 'armed' spider Phoneutria nigriventer, Toxicon, 29 (1991) 1225-1233. [16] Romano-Silva, M.A., Gomez, M.V. and Brammer, M.J., Modulation of Ca2+-stimulated glutamate release from synaptosomes by Na+ entry through tetrodotoxin-sensitive channels, Biochem. J., 304 (1994) 353-357. [17] Wessler, I., Dooley, D.J., Werland, J. and Sclemmer, F., Differential effects of calcium channel antagonists (w-conotoxin GVIA, nifedipine, verapamil) in the electrically-evoked release of [3H]acetylcholine from the myenteric plexus, phrenic nerve and neocortex of rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 341 (1990) 288-294.